Chapter 1, The Reptilian Spectacle - UWSpace - University of ...

4.2 Methods & Materials
4.2.1 Sample Collection and Solubilization
Shed skins were collected from snakes (Masticophis flagellum, Elaphe guttata) and geckos (Phelsuma
madagascariensis grandis, Gekko marmoratus) kept on site as well as from zoos and private pet
owners (all other species). The spectacle scales from most sheds were used in the study of spectral
transmission described in Chapter 3. Scales of interest were cut out, taking care to remove all
surrounding material to ensure only the hard scales were used, with the exception of gecko dorsal
scales which are too small to individually excise. Scales were briefly washed in turn in distilled water
and 100% ethanol, ensuring that all debris was removed, and allowed to air dry. They were then placed
in a solubilization buffer consisting of 7 M urea, 2 M thiourea, 25 mM tris-HCl (pH 8.5), and 5% ß-
mercaptoethanol. Protease inhibitors were omitted due to thiourea’s significant antiproteolytic property
at concentrations of 2 M (Castellanos-Serra & Paz-Lago, 2002). Samples were left to digest for 12
hours at room temperature, then pulse sonicated on ice until visibly broken into smaller pieces, and
again left at room temperature for 36 hours before being centrifuged and the supernatant collected and
frozen at -80ºC. This method combines characteristics of two other established methods for keratin
extraction. The recipe (the 2 M thiourea in particular) and 48 hour extraction time were inspired by the
Shindai Method (Nakamura et al., 2002) for extracting alpha keratins from human hair, while the
higher urea content, mechanical pulverization, and room temperature digestion were adopted from
methods used in the extraction of α and ß keratins from snake integument (Toni & Alibardi 2007a, who
cite Sybert et al., 1985, as the source of their method).
Protein concentrations were measured using a Bradford colorimetric assay (Bio-Rad Protein
Assay, Bio-Rad) on samples diluted between 10-100 times with double-distilled water, which not only
brought the protein concentrations to within the linear range of the assay, but also reduced the urea
concentrations to levels that would not interfere with its accuracy.
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